Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motoneurons. It affects 500,000 individuals worldwide annually and remains untreatable. To develop an effective treatment for ALS, a precise understanding of its pathogenesis is indispensable. One approach toward this goal involves identifying the gene(s) responsible for familial forms of ALS. Such an approach has uncovered mutations in the gene encoding Cu/Zn cytosolic superoxide dismutase (SOD1); these mutations only account for 20%-25% of familial cases. Despite intense efforts, the pathogenesis of the remainder of familial cases and all of sporadic cases are practically unknown. We have recently identified a new NMDA type glutamate receptor, NR3B. Interestingly, it is expressed almost exclusively in the motoneurons known to be affected in ALS and negatively regulates the function of NMDA receptor to limit Ca2+ influx. Because it is well documented that an overactivation of NMDA receptor causes the cell death associated with various neurological disorders, the identification of NR3B lead us to propose the "NR3B hypothesis" of the pathogenesis of ALS. This hypothesis proposes that defects in NR3B function may be neurotoxic in motoneurons through loss of the negative regulation of motoneuronal NMDA-mediated Ca2+ currents. Loss of NR3B function may increase the NMDA receptor current and Ca2+ influx in these cells, thereby predisposing motoneurons to excitotoxicity. This mechanism may cause ALS by itself or may predispose carrier to ALS when it is combined other genetic or epigenetic factors. In this proposal, we will test this NR3B hypothesis by genetic analysis of the NR3B gene in humans and modifications of NR3B gene dose in ALS mice. We will first analyzing DNA from ALS patients for mutations in the NR3B gene. We will first screen ALS families without a known genetic defect for genetic linkage to the NR3B gene (Aim 1). We will then perform mutational analysis of the full NR3B gene in linked cases and in a series of individuals with both familial and sporadic ALS, as well as controls (Aim 2). Thereafter (Aim 3) we will test the hypothesis that polymorphisms in the NR3B gene are associated with an increased risk of developing ALS or with some aspect of the clinical course of ALS (e.g. onset age, life span after onset, disease distribution). In related studies in mice, we will determine whether variations in the dose of the NR3B gene alter the phenotype of transgenic SOD1-G93A mice (Aim 4). In our view, this is a significant translational and explorative investigation that promises to relate a newly discovered, novel motoneuronal glutamate receptor subunit, NR3B, with a fatal human motor neuron disease; the findings have potential implications for the development of new approaches to therapy in ALS.